The present invention is in the field of plant genetics and relates to improved methods for mutagenesis, gene identification and analysis of gene function in crop plants. The methods are useful in any plant species and their use in tomato is exemplified herein.
The genomes of higher plants are estimated to contain 30,000 to 50,000 genes. A function has been ascribed to only a few hundred plant genes. The isolation of new genes, and the mutation of these newly isolated genes, is frequently required to ascertain gene function. Crop improvement through biotechnology depends on detailed characterization of newly isolated genes.
The Arabidopsis model system has greatly contributed to the remarkable advances in plant molecular biology during the last decade. The major reasons for the successful use of Arabidopsis are its small size, short life cycle and relatively small genome (Leutwiler et al., 1984). Additionally, Arabidopsis can be easily transformed with foreign DNA (Bechtold et al., 1993). These features facilitate the genetic dissection of any trait expressed in Arabidopsis through screening of large populations of mutants for the various genes, which control a trait of interest. Plant populations mutagenized by ethyl methanesulfonate (EMS), fast neutron bombardment, T-DNA insertions, and transposon tagging have proved invaluable to plant biologists as a means of dissecting the genetic control of plant development and genome traits (Koncz et al., 1992). Despite the considerable advantages of using Arabidopsis as a model for genetic analysis, it is not a crop plant, and the knowledge acquired in this species cannot always be applied to other agronomically important crop species. For example, Arabidopsis has a silique type of fruit and therefore it is a good model species for fruit development in members of the Brissicaceae but is not useful for plants which produce a fleshy, berry-type, fruit
On the other hand, tomato (Lycopersicon esculentum) is a good model for crop species that produce a fleshy, berry-type fruit. Tomato is well known genetically. Tomato has a relatively small diploid genome (n=12, C=1 pg) containing hundreds of mapped traits and molecular markers (Tanskley, 1993). Tomato can be transformed with foreign DNA (McCormick et al., 1986). Moreover, it is one of the most important crops in the fresh vegetable market as well as in the food processing industry (Hille et al., 1989; Rick and Yoder, 1988).
A major obstacle to making further advances in tomato genetics is the lack of large mutant populations required for gene identification. A useful mutant population for tomatoes would contain at least one mutant allele for every tomato gene. Such a population would make it possible to achieve saturated mutagenesis in this crop. Although techniques exist for producing mutant tomato plants, it is currently impractical, due to time and space constraints, to apply these techniques on a sufficiently large scale to obtain populations in which the genome is saturated with mutations. These same constraints limit research in other agronomic crops.
Mutant tomato plants have been produced through the use of DNA damaging agents such as EMS (Hildering and Verkerk, 1965; Schoenmakers et al., 1991; Wisman et al., 1991), X-rays (Hildering and Verkerk, 1965), or fast-neutrons (Verkerk, 1971), although to a much more limited extent compared to similar efforts in Arabidopsis. A few hundred mutant tomato lines, available through the Tomato Genetic Resource Center, have been described, but no stocks of mutagenized M2 seeds, originating from a large population of M1 plants, are available for screening mutations in new genes.
Insertional mutagenesis by T-DNA tagging is not practical in tomato as transformation procedures are still laborious. Transposon tagging, on the other hand, is a promising approach for mutagenesis and gene identification in tomato and other agronomic species. The Ac/Ds transposable element family has been shown to be active in tomato (Yoder et al., 1988) and patterns of Ac/Ds transposition in this species have been described (Carroll et al., 1995; Osborne et al., 1991; Rommens et al., 1992; Yoder et al., 1988). Tomato lines have been produced containing Ds elements that were mapped in the tomato genome (Knapp et al., 1994; Thomas et al., 1994). These lines make it possible to take advantage of the preferential insertion of Ac/Ds at nearby sites (Dooner and Belachew, 1989; Jones et al., 1990). The Ac/Ds tagging system was used to tag and isolate several genes, such as cf9, a locus responsible for Cladosporium resistance (Jones et al., 1994); dwarf, a gene encoding a cytochrome p450 homolog (Bishop et al., 1996); and dcl which controls chloroplast development (Keddie et al., 1996).
Reverse genetics is an efficient strategy for determining the function of an isolated gene (Benson et al. 1995). In maize, for example, a mutation in a gene of interest can be identified by screening a large plant population composed of 48,000 randomly mutagenized plants. In principle, each plant in this mutant population contains a different mutation caused by insertion of a transposable element. A plant containing the insertion of a transposable element in the gene of interest is identified by polymerase chain reaction (PCR) analysis. A first primer having a nucleotide sequence corresponding to the transposon, and a second primer having a nucleotide sequence corresponding to the gene of interest, are used in the PCR reaction with DNA isolated from presumptive mutants. In principle, a PCR product is only produced if the transposon is inserted in the gene of interest. Mutant plants comprised of DNA from which a PCR product is produced in the PCR reaction are analyzed to determine the effect of the mutation on plant growth and development and the function of the gene of interest is thereby ascertained.
It is impractical to use reverse genetics in most crop species, however, because it would require considerable time and effort, and extensive field facilities, to produce and accommodate the tens of thousands of T-DNA or transposon-tagged plants that must be grown to maturity to detect the mutant of interest. Accordingly, an alternative strategy is required to make reverse genetics a reality in most crop species. Likewise, a practical method is required to screen large populations of crop plants transformed with a DNA construct capable of detecting a DNA element which controls gene expression such as a promoter or an enhancer.
It is an object of the present invention to provide improved methods for mutant identification and characterization using a miniaturized crop plant.
It is another object of the present invention to provide improved methods for characterization of cloned nucleotide sequences.
It is yet another object of the present invention to provide improved methods for the cloning of nucleotide sequences.
These objects, and others, are achieved by providing a method for selecting a mutant miniature plant having a desired trait, comprising the steps of:
(a) providing a population of miniature plants, wherein said miniature plants have the following characteristics: (i) reduced size in comparison to a commercial cultivar of the same species; (ii) maturation to produce viable seeds or tubers at a plant density of at least ten-fold higher than standard growth conditions used for a commercial plant of the same species; and (iii) capable of being crossed with a commercial plant of the same species;
(b) generating mutant miniature plants in said miniature plant population by treating said miniature plants with a mutation-inducing agent, to produce a mutant plant population; and
(c) selecting a mutant miniature plant having said desired trait within said mutagenized miniature plant population.
In all aspects and embodiments of the present invention as described herein, the population of miniature plants may be generated by natural or induced mutations, by genetic engineering, or by treatment with plant growth factors. Examples of miniature plants that can be used according to the invention include, but are not limited to, miniature tomato cultivars such as xe2x80x98Micro-Tomxe2x80x99 and xe2x80x98Micro-Peachxe2x80x99. The mutation-inducing agent used in step (b) above may be a chemical mutagen such as ethyl methanesulfonate (EMS), methyl methane-sulfonate (MMS), methyl-N-nitrosourea (MNU), and bleomycins. Alternatively, the mutation-inducing agent may be irradiation such as UV, xcex3-irradiation, X-rays, and fast neutrons. Finally, the mutation-inducing agent may be a mobile DNA sequence which is a T-DNA or a transposable element which is selected from the group consisting of an autonomous transposon, a non-autonomous transposon, and an autonomous/non-autonomous transposon system such as, but not being limited to, the maize Ac/Ds transposable element. The commercial plant of the same species is a plant used to produce food, fiber or flowers, including but not being limited to, plants which produce a berry-type fruit such as tomato, grape, prune, eggplant, citrus fruits, and apple, or a plant of the Solanaceae family, e.g. potato.
In another embodiment, the present invention provides a mutant miniature population wherein a miniature plant of said population has the following characteristics: (i) reduced size in comparison to a commercial plant of the same species; (ii) matures to produce viable seeds or tubers at a density of at least ten-fold higher than standard growth conditions used for a commercial cultivar of the same species; (iii) capable of being crossed with a commercial plant of the same species; and (iv) carries a mutation induced by an agent which is a chemical mutagen, irradiation, or a mobile DNA sequence.
Yet another embodiment of the present invention provides a method for identifying a miniature plant containing a mobile DNA sequence inserted into a gene of interest comprising the steps of:
(a) providing a population of miniature plants, wherein said miniature plants have the following characteristics: (i) reduced size in comparison to a commercial plant of the same species; (ii) maturation to produce viable seeds or tubers at a plant density of at least ten-fold higher than standard growth conditions used for a commercial plant of the same species; and (iii) capable of being crossed with a commercial plant of the same species;
(b) generating mutant plants in said population of miniature plants by treating said plants with a mobile DNA sequence;
(c) screening DNA extracted from said mutant plants by PCR using a first primer to a nucleotide sequence corresponding to said mobile DNA sequence and a second primer corresponding to a nucleotide sequence of said gene of interest; and
(d) identifying a miniature plant comprised of DNA which produces a PCR product in the presence of said fist and second primers.
Yet another embodiment of the present invention provides a method for producing a mutant population of a miniature plant comprising the steps of:
(a) providing a population of miniature plants, wherein said miniature plants have the following characteristics: (i) reduced size in comparison to a commercial plant of the same species; (ii) maturation to produce viable seeds or tubers at a plant density of at least ten-fold higher than standard growth conditions used for a commercial plant of the same species; and (iii) capable of being crossed with a commercial cultivar of the same species; and
(b) generating said mutant plants in said miniature plant population by treating said miniature plants with a mutation-inducing agent.
When said mutation-inducing agent of step (b) is a T-DNA, the miniature plants are infected with Agrobacterium, thus producing multiple transformants wherein each transformant contains a T-DNA insertion in a different genomic position. When said mutation-inducing agent of step (b) is a transposon, the mutant miniature plant population is obtained from the progeny of miniature plants containing an active transposition system. This active transposition system may be a plant native transposon or a transposon introduced into the plant by genetic engineering techniques well known to an artisan in the field, such as an autonomous transposon or a transposable element obtained by crossing a plant containing a non-autonomous transposon with either a transposase source or a plant containing an autonomous transposon. The transposable element is, for example, the maize Ac/Ds transposon system.
Yet another embodiment of the present invention provides a method for identifying a nucleotide sequence which controls plant gene expression comprising the steps of:
(a) transforming a miniature plant of a crop plant with a DNA construct to produce a population of randomly mutagenized plants, wherein said DNA construct comprises a gene sequence encoding a screenable marker which lacks a promoter or contains a minimal promoter, wherein said miniature plant has the following characteristics: (i) reduced size in comparison to a commercial plant of the same species; (ii) maturation to produce viable seeds or tubers at a plant density of at least ten-fold higher than standard growth conditions used for a commercial plant of the same species; and (iii) capable of being crossed with a commercial cultivar of the same species to produce a population of randomly mutagenized plants;
(b) identifying a miniature plant within said plant population which is transformed with said DNA construct and expresses said screenable marker; and
(c) cloning the nucleotide sequence which is operably linked to said gene encoding said screenable marker from the total DNA isolated from said transformed miniature plant identified in step (b).
The screenable marker may be GUS or luciferase, the mobile DNA sequence may be a T-DNA or a transposable element and the nucleotide sequence which controls plant gene expression may be a promoter or an enhancer.
In yet a further embodiment, the invention provides a method for producing a mutant population of a commercial plant with a desired trait, which comprises the steps of:
(a) crossing a mutant miniature plant having said desired trait selected according to the selection method of the present invention, with a commercial plant of the same species; and
(b) selecting progeny which resemble the commercial parent plant and express said desired trait.
According to this embodiment, the invention also encompasses mutant populations of commercial plants obtained by the above method.